Project Summary/Abstract In a multicellular organism, every decision and action taken by a cell depends on communication with its neighbors. Lethal diseases, such as cancer, can arise when normal communication channels are disrupted. In this proposal, we investigate two specialized communication protocols that serve in the exchange of complex information packets between participating cells. In the first type, cells package proteins and genetic material into tiny, membrane-encapsulated containers, called vesicles, for delivery to recipient cells. In the second protocol, cells extend long and thin membrane tubules that form highways between participating cells for free or regulated exchange of cellular contents. Our central hypothesis is that these two important forms of intercellular communication are regulated by sugary polymers that cells assemble on their outer membrane. Like a compressed gas hovering over the cells, we propose that these sugary polymers can generate a pressure that makes it easier to bend the membrane into the spherical and tubular forms required for vesicles and intercellular highways. Thus, we anticipate that cells can ramp up communication by assembling more sugary polymers on the cell surface, or, conversely, suppress communication through a reduction of cell-surface polymers. In this proposal, our aims are to (1) determine how and what type of information is exchanged through the membrane bridges; (2) identify how the formation of the membrane bridges are controlled by the internal cellular skeleton and its regulators; and (3) determine the optimal conditions for vesicle generation and transfer of messages to participating cells. To study these possibilities, we will use sophisticated new imaging techniques capable of resolving ultrasmall cellular features, like the membrane structures that are under investigation here. We also take advantage of our ability to create DNA instruction sets that can program cells to assemble new and different polymer types on their outer membrane. A major goal is to identify the types of messages that are sent from donor to receiver cells. In addition to advanced imaging approaches, we will use powerful, ?next-generation? techniques that can simultaneously identify large numbers of proteins or nucleic acids (i.e. genetic instructions), which may be part of the messages transferred. The new understanding that we seek to develop should have broad relevance in biomedicine. In particular, aggressive cancer cells often produce and attach unusual numbers of sugary polymers on their outer membrane. Thus, our studies could provide new insight into how intercellular communication goes awry in cancer, and how we might intervene therapeutically to normalize and correct the flow of information among our cells.